Project Summary Scientists can create thousands of chemically distinct nanoparticles using a growing number of high throughput chemistries, but it is still difficult to test more than a few nanoparticles in vivo. The goal of this work is to substantially improve how lipid nanoparticles (LNPs) deliver nucleic acid therapies by performing a systematic high throughput in vivo LNP study. This goal will be achieved using cutting edge DNA barcoded nanoparticles; deliverer mediated by 300 different nanoparticles can be measured in a single mouse. 4,320 chemically distinct nanoparticles will be tested in vitro and in vivo, focusing on 2 fundamental questions. First, how does nanoparticle structure affect cell targeting in vivo? Nanoparticle chemical and physical traits affect delivery in vitro. However, the extent to which the same LNP traits influence delivery in animals (in vivo) is unclear. A recently developed bioinformatics pipeline will be used to (i) systematically analyze how LNP structure affects in in vivo delivery in macrophages, endothelial cells, and hepatocytes, both in vitro and in vivo. The same data will be used to (ii) quantify the precision with which in vitro drug delivery predicts in vivo drug delivery. Second, how do clinically relevant physiological changes affect delivery in vivo? LNPs are similar to lipoproteins, which are natural lipid-containing nanostructures. Lipoproteins are actively trafficked to endothelial cells, macrophages, and hepatocytes in vivo. Given that lipoprotein trafficking changes in patients with high cholesterol, taking statins, and patients with many other conditions, LNP transport may also change. The top 600 in vivo LNPs from the 4,320 LNP in vivo screen will be administered to genetic mouse models of aberrant lipid transport in order to (iii) investigate how genetic alterations in cholesterol trafficking affect in vivo delivery. This work will make 5 significant contributions to nanotechnology. First, the extent to which LNP chemical traits influence delivery directly in vivo will be tested; relationships between nanoparticle structure and delivery are studied in vitro. Second, the precision with which in vitro nanoparticle delivery predicts in vivo delivery will be quantified. This could increase the efficiency with which clinical nanoparticles are discovered. Third, the effect of clinically relevant physiological changes on LNP delivery will be examined. Nanoparticles can interact with cholesterol trafficking pathways; these interactions are likely to change with disease and can affect nanoparticle targeting / safety. Fourth, the feasibility of studying thousands of LNPs in vivo will be demonstrated. Fifth, open source protocols for nanoparticle barcoding will be established and disseminated. These results will provide crucial insight into the ways LNP chemical traits and specific genes alter LNP delivery, informing the design of LNPs that deliver nucleic acid cargos (e.g., siRNA, mRNA, CRISPR-Cas9) for numerous therapeutic applications.